Written by Miyako Rogers, Science Writer
New research has shown that existing methods of circulating tumour cell (CTC) analysis exclude mesenchymal CTCs, which may have prognostic value. This study introduces a new method of CTC analysis to identify and isolate CTCs missed by standard CTC categorisation using microfluidics and single-cell genomics analysis. This epitope-independent method of CTC analysis could improve the prognostic power of liquid biopsies in non-small cell lung cancer (NSCLC).
Using liquid biopsies in NSCLC
Lung cancer is the leading cause of cancer mortality worldwide, causing an estimated 1.8 million deaths in 2020 alone. Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases and is usually diagnosed at advanced stages, with an estimated 5-year survival rate of 26.4%. Despite advances in cancer treatment such as immunotherapy, most patients either do not respond to treatment at all or develop resistance later on.
For the diagnosis of NSCLC, lung biopsies are invasive, complex and can lead to complications and long recovery times. Circulating tumour cells (CTCs) present an opportunity to perform liquid biopsies, which are minimally invasive as they only require a small blood sample. CTCs are tumour-derived cells, which are shed into the bloodstream. As a result, they mirror the genetic makeup of primary tumours and their metastases (secondary growths), which makes them excellent candidates for diagnostics. Furthermore, it is difficult to obtain repeat biopsies from tumours, so CTCs could be a source of information on the genetic changes occurring in tumours over the course of treatment.
A new method of CTC analysis
Previous CTC analysis focussed on cells expressing epithelial markers, but epithelial-to-mesenchymal transition (EMT) is a key step in tumour metastasis. During this process, epithelial markers are downregulated, and so may be missed by this form of CTC isolation. To overcome this issue, the researchers, combined microfluidics, immunofluorescence and single-cell genomics analysis to identify and isolate CTCs which were often likely missed by standard CTC categorisation.
Microfluidic devices allow for epitope-independent isolation of CTCs as they sort cells based on size; CTCs are larger in size and less likely to deform than normal blood cells. Combining microfluidic enrichment with immunofluorescence staining allowed for the categorisation of vimentin-positive CTCs. Vimentin is a well-documented marker for cancer, but to doubly make sure these cells were cancerous, they also performed whole-genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) on a subset of isolated CTCs. Single-cell copy number alteration (CNA) analysis was then performed to identify genomic alterations to confirm the cancerous nature of these CTCs.
In conclusion, this study reveals that a large proportion of CTCs, which potentially have prognostic value, are commonly missed by standard CTC categorisation. Furthermore, this research advances the potential of liquid biopsies as a diagnostic tool and showcases the potential for microfluidic enrichment and single-cell genomics in CTC categorisation.
Image Credit: Canva
